Uncoupling of muscle-specific protein expression in myocyte X myoblast heterokaryons

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat α-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.     

Uncoupling protein 1: a short-circuit in the chemiosmotic process

One of the significant naturally occurring short-circuits in the chemiosmotic process is manifest through the activity of uncoupling protein 1, most notably present in the inner membrane of mitochondria in brown adipose tissue. Much has been learnt about the mechanism of action of uncoupling protein 1 since its discovery in the 1970’s, yet many areas of controversy surround (i) the mechanism of its action, (ii) novel regulators of its activity, and (iii) the tissue specificity of its expression. In this mini-review, areas of controversy are highlighted from the aforementioned categories using selected key experiments to demonstrate the arguments.     

Response of rat exocrine pancreas to high-fat and high-carbohydrate diets

Intake of diets with high fat content is a risk factor for acute pancreatitis and pancreatic cancer. The underlying mechanisms leading to the development of these diseases due to high fat intake are currently unknown. The current study was designed in rats to determine the physiologic and pathological consequences of a highfat diet that contained excess amounts of cottonseed oil or a high-carbohydrate diet that contained high amounts of sucrose on the exocrine pancreas. Rats were maintained on the diets for 4 weeks, and a cannula was inserted into the right jugular vein and one into the pancreatic duct for collection of pancreatic juice. Volume of the pancreatic juice and concentrations of amylase, lipase, and trypsinogen in the pancreatic juice were measured before and after infusions of CCK-8. Results showed that basal and CCK-stimulated pancreatic outputs of volume, amylase and lipase but not trypsinogen, were significantly elevated in intact rats given a high-fat diet when compared with rats given a high-carbohydrate diet. Forty-eight hours later, rats were sacrificed, and parts of the pancreas were removed for isolation of pancreatic acinar cells and for histopathologic studies. Pancreatic acini isolated from rats on a high-fat diet showed significantly lower basal and CCK-stimulated amylase release when compared with those on a high-carbohydrate diet. Histology of the pancreas of rats on a high-carbohydrate diet appeared normal; however, the pancreas of rats on high-fat diet showed significant alterations in exocrine pancreas. These results showed abnormalities in the exocrine pancreas of rats on a high-fat diet, that were not found in rats on a high-carbohydrate diet; further, they support the contention that a high-fat diet has a deleterious effect on the pancreas.     

Uncoupling mechanism and redox regulation of mitochondrial uncoupling protein 1 (UCP1)

Brown adipose tissue (BAT) and brown in white (brite) adipose tissue, termed also beige adipose tissue, are major sites of mammalian nonshivering thermogenesis. Mitochondrial uncoupling protein 1 (UCP1), specific for these tissues, is the key factor for heat production. Recent molecular aspects of UCP1 structure provide support for the fatty acid cycling model of coupling, i.e. when UCP1 expels fatty acid anions in a uniport mode from the matrix, while uncoupling. Protonophoretic function is ensured by return of the protonated fatty acid to the matrix independent of UCP1. This mechanism is advantageous for mitochondrial uncoupling and compatible with heat production in a pro-thermogenic environment, such as BAT. It must still be verified whether posttranslational modification of UCP1, such as sulfenylation of Cys253, linked to redox activity, promotes UCP1 activity. BAT biogenesis and UCP1 expression, has also been linked to the pro-oxidant state of mitochondria, further endorsing a redox signalling link promoting an establishment of pro-thermogenic state. We discuss circumstances under which promotion of superoxide formation exceeds its attenuation by uncoupling in mitochondria and throughout point out areas of future research into UCP1 function.     

Uncoupling protein 1 affects the yeast mitoproteome and oxygen free radical production

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.     

Differential body weight and feeding responses to high-fat diets in rats and mice lacking cholecystokinin 1 receptors

Prior data demonstrated differential roles for cholecystokinin (CCK)1 receptors in maintaining energy balance in rats and mice. CCK1 receptor deficiency results in hyperphagia and obesity of Otsuka Long-Evans Tokushima Fatty (OLETF) rats but not in mice. To ascertain the role of CCK1 receptors in high-fat-diet (HFD)-induced obesity, we compared alterations in food intake, body weight, fat mass, plasma glucose, and leptin levels, and patterns of hypothalamic gene expression in OLETF rats and mice lacking CCK1 receptors in response to a 10-wk exposure to HFD. Compared with Long-Evans Tokushima Otsuka (LETO) control rats, OLETF rats on HFD had sustained overconsumption over the 10-wk period. High fat feeding resulted in greater increases in body weight and plasma leptin levels in OLETF than in LETO rats. In situ hybridization determinations revealed that, while HFD reduced neuropeptide Y (NPY) mRNA expression in both the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH) of LETO rats, HFD resulted in decreased NPY expression in the Arc but not in the DMH of OLETF rats. In contrast to these results in OLETF rats, HFD increased food intake and induced obesity to an equal degree in both wild-type and CCK1 receptor(-/-) mice. NPY gene expression was decreased in the Arc in response to HFD, but was not detectable in the DMH in both wild-type and CCK1 receptor(-/-) mice. Together, these data provide further evidence for differential roles of CCK1 receptors in the controls of food intake and body weight in rats and mice.     

Stimulation of microperoxisomal beta-oxidation in rat heart by high-fat diets

1. Heart microperoxisomal beta-oxidation activity, measured as cyanide-insensitive palmitoyl-CoA-dependent NAD+-reduction, was detected in a microperoxisome-enriched fraction from rat myocardium. The effect on this microperoxisomal beta-oxidation of the fatty acid composition of the dietary oils was investigated. 2. Feeding 15% (w/w) high erucic acid rapeseed oil or partially hydrogenated marine oil for 3 weeks increased the microperoxisomal beta-oxidation in the heart 4-5-fold, compared to a soybean oil diet. Increasing amounts (5-30%, w/w) of partially hydrogenated marine oil in the diet led to a 3-fold increase in the microperoxisomal beta-oxidation capacity at 20% or more of this oil in the diet. 3. The activity of the microperoxisomal marker enzyme catalase followed closely the cyanide-insensitive palmitoyl-CoA-dependent NAD+-reduction, except when feeding more than 20% (w/w) partially hydrogenated marine oil where a significant decrease in the catalase activity was observed. 4. In rapeseed oil-fed animals the extent of increase of microperoxisomal beta-oxidation was directly correlated to the amount of erucic acid (22:1, n-9 cis) in the diet. 5. Feeding partially hydrogenated rapeseed oil or partially hydrogenated soybean oil resulted in activities of microperoxisomal beta-oxidation significantly lower than in the corresponding unhydrogenated oils. No significant difference could be detected between diets containing hydrogenated or unhydrogenated marine oil. 6. Addition of 5% soybean oil to the essential fatty acid-deficient, partially hydrogenated marine oil diet did not change the effect on the microperoxisomal beta-oxidation activity. 7. Clofibrate feeding increased the heart microperoxisomal beta-oxidation capacity 2.5-fold, as compared to a standard pelleted diet. 8. These findings are discussed in relation to the transient nature of the cardiac lipidosis observed with animals fed on diets rich in C22:1 fatty acids. It is concluded that the heart plays an important part in the adaptation process.     

Butyrylcholinesterase activity in plasma of rats and rabbits fed high-fat diets.

1. Comparative studies with rats and rabbits were carried out to address the question as to whether the amount of dietary fat affects butyrylcholinesterase (EC 3.1.1.8.) activity in plasma. 2. Plasma butyrylcholinesterase activities were about 5-fold higher in rabbits than rats. 3. Ad libitum feeding of diets enriched with corn oil caused increased body weights in rabbits but not in rats 4. Plasma butyrylcholinesterase activities of rats were increased with increasing intakes of corn oil. In rabbits, such an effect could not be demonstrated conclusively. 5. Evidence is presented that in rats the substitution of dietary corn oil for isocaloric amounts of either carbohydrates or protein produces similar increases in plasma butyrylcholinesterase activity. 6. This suggests that among macronutrients the amount of fat primarily determines butyrylcholinesterase activity in the plasma of rats.     

Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum fibroblast growth factor-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.     

Effect of high-fat diet, surrounding temperature, and enterostatin on uncoupling protein gene expression

Nonshivering thermogenesis induced in brown adipose tissue (BAT) during high-fat feeding is mediated through uncoupling protein 1 (UCP1). UCP2 is a recently identified homologue found in many tissues. To determine the role of UCP1 and UCP2 in thermoregulation and energy balance, we investigated the long-term effect of high-fat feeding on mRNA levels in mice at two different ambient temperatures. We also treated mice with the anorectic peptide enterostatin and compared mRNA levels in BAT, white adipose tissue (WAT), stomach, and duodenum. Here, we report that high-fat feeding at 23 degrees C increased UCP1 and UCP2 levels in BAT four- and threefold, respectively, and increased UCP2 levels fourfold in WAT. However, at 29 degrees C, UCP1 decreased, whereas UCP2 remained unchanged in BAT and increased twofold in WAT. Enterostatin increased UCP1 and decreased UCP2 mRNA in BAT. In stomach and duodenum, high-fat feeding decreased UCP2 mRNA, whereas enterostatin increased it. Our results suggest that the regulation of uncoupling protein mRNA levels by high-fat feeding is dependent on ambient temperature and that enterostatin is able to modulate it.     

Induction of peroxisomal beta-oxidation in rat liver by high-fat diets.

Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.     

A high-fat, refined-carbohydrate diet affects renal NO synthase protein expression and salt sensitivity.

Chronic consumption of a high-fat, refined-carbohydrate (HFS) diet causes hypertension. In an earlier study, we found increased nitric oxide (NO) inactivation by reactive oxygen species (ROS) and functional NO deficiency in this model. Given the critical role of NO in renal sodium handling, we hypothesized that diet-induced hypertension may be associated with salt sensitivity. Female Fischer rats were fed an HFS or a standard low-fat, complex-carbohydrate (LFCC) rat chow diet starting at 2 mo of age for 2 yr. Arterial blood pressure, renal neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), and inducible NO synthase (iNOS) protein and nitrotyrosine abundance (a marker of NO inactivation by ROS), and urinary NO metabolite excretion were measured. To assess salt sensitivity, the blood pressure response to a high-salt (4%) diet for 1 wk was determined. After 2 yr, renal nNOS and urinary NO metabolite excretion were significantly depressed, whereas arterial pressure, eNOS, iNOS, and nitrotyrosine were elevated in the HFS group but remained virtually unchanged in the LFCC group. Consumption of the high-salt diet resulted in a significant rise in arterial pressure in the HFS, but not in the LFCC, group. Thus chronic consumption of an HFS diet results in hypertension and salt sensitivity, which may be in part due to a combination of ROS-mediated NO inactivation and depressed renal nNOS protein expression.     

Uncoupling protein and alternative oxidase of Dictyostelium discoideum: occurrence,properties and protein expression during vegetative life and starvation-induced early development

In this study we show that mitochondria of Dictyostelium discoideum contain both alternative oxidase (AOX) and uncoupling protein (UCP). AOX was stimulated by purine mononucleoside and was monomeric. UCP was stimulated by free fatty acids and was poorly sensitive to GTP. Both proteins collaborated in energy dissipation when activated together. AOX expression in free-living ameboid cells decreased strongly from exponential to stationary phase of growth but much less during starvation-induced aggregation. In contrast, UCP expression was constant in all conditions indicating permanent need. Our results suggest that AOX could play a role in cell differentiation, mainly by protecting prespore cells from programmed cell death.     

解偶联蛋白2与衰老

解偶联蛋白(uncoupling protein,UCP)属于线粒体内膜上的一类载体蛋白,其生理作用是消除线粒体膜电位,使氧化磷酸化解偶联,从而抑制酸腺苷(adenosine triphosphate,ATP)合成,能量以热能形式散发.研究发现UCP2具有一种质子漏功能,表现对线粒体活性氧(reactive oxygen species,ROS)产生的调控和降低ROS的功能.在不同组织器官,不同代谢状态下UCP2的生理功能对细胞的影响不完全相同.特别是近年来的研究发现,UCP2参与了能量代谢、ROS的产生、子宫内膜退化、衰老等过程,并且与非酒精性脂肪肝、抗肥胖、动脉粥样硬化、局部缺血以及缺血再灌注损伤和2型糖尿病等有一定的相关性,倍受人们的关注.     

Uncoupling of 3T3-L1 gene expression from lipid accumulation during adipogenesis

Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.     

Uncoupling protein-1 mRNA expression in lipomas from patients bearing pathogenic mitochondrial DNA mutations

Multiple symmetric lipomatosis (MSL) is a rare disorder characterised by large subcutaneous fat masses in some parts of the trunk. Mitochondrial disfunction is common in MSL, but the identity of the adipose cells developing in multiple lipomas is not well known. We determined that brown adipose tissue-specific uncoupling protein-1 (UCP-1) mRNA is expressed in the lipoma of a multiple symmetric lipomatosis patient bearing the 8344 mutation in the tRNALys gene of mitochondrial DNA. UCP1 mRNA was not detected in normal subcutaneous fat from the same patient or in the lipoma of another patient bearing a different mutation in the same tRNALys gene. These findings implicate brown adipose cells as the origin of lipomas in a subset of patients bearing tRNALys mutations in mitochondrial DNA.